1. Volume 18, Issue 8, Pages 1011-1021 (August 2010)
Mapping the Interactions between a Major Pollen Allergen and Human IgE Antibodies 
Guilherme Razzera, Gabriele Gadermaier, Viviane de Paula, Marcius S. Almeida, Matthias Egger, Beatrice Jahn-Schmid, Fabio C.L. Almeida, Fatima Ferreira, Ana Paula Valente 
Structure 
Volume 18, Issue 8, Pages (August 2010)
DOI: /j.str
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2. Figure 1
NMR Solution Structure of the mugwort Pollen Major Allergen Art v 1
(A) Backbone superposition of 20 lowest energy Art v 1 structures. The defensin domain is shown in dark blue, the intermediate region in blue, and the C-terminal part in light blue.
(B) In the ribbon illustration, disulphide pairings Art v 1 are indicated by numbers and yellow sticks (PDB ID code 2kpy).
Structure  , DOI: ( /j.str )
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3. Figure 2 Backbone Dynamics of Art v 1
(A) Heteronuclear NOE data reveal three regions of Art v 1 movements. Proline residues are represented by red asterisks.
(B) Pico-nanosecond timescale backbone dynamics are represented by order parameters (S2).
(C) Milli-microsecond chemical exchange values [Rex(s-1)] are plotted as bars as a function of the residue number.
(D) Art v 1 residues are colored according to their order parameter (S2): (i) blue in the well-folded defensin domain, (ii) purple in the transitional region, and (iii) red in the C terminus. Prolines and nonobserved residues are indicated in gray. Residues involved in conformational exchange are highlighted by numbers in the structure. Error estimates were calculated by the program Modelfree.
Structure  , DOI: ( /j.str )
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4. Figure 3
NMR Analysis of Natural Art v 1 Based on Natural Abundance 13C-HSQC Spectra
(A) Chemical shift deviations between natural and recombinant Art v 1 α-hydrogen and carbons are shown for individual amino acids. The average (Δδ) and one standard deviation from the average (Δδ + σ) cutoffs are represented by orange and red dashed lines, respectively. Bars for corresponding residues are shown in the same color; prolines are represented as gray bars.
(B) Shifted amino acids are highlighted in orange (>Δδ) and red (>Δδ + σ) in the structure of Art v 1 (2kpy).
(C) Resonances in the Hα/Cα region of the 1H/13C HSQC spectrum are illustrated in black for the recombinant protein and in red for the natural counterpart. Proline resonances (highlighted by the dotted box) clearly changed but could not be assigned by comparison with values from the recombinant protein.
Structure  , DOI: ( /j.str )
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5. Figure 4 Art v 1 IgE Epitopes
HSQC of 15N labeled rArt v1 were acquired in the absence and presence of specific human IgE purified from the peripheral blood of a mugwort pollen-allergic patient. All experiments were measured in 50 mM citrate buffer (pH 7.8).
(A) For distinct amino acids chemical shift perturbations (CSP) observed upon IgE interaction.
(B) Sections of the actual spectrum showing changes in specific amino acids, in black free Art v1 and in red in the presence of IgE.
(C and D) Residues with high CSP (>Δδ + σ) are represented in red; those with lower CSP (>Δδ) are in orange.
(E) The charge distribution on the Art v 1 surface is depicted in blue and red for positive and negative charges, respectively. Residues 71–108 displaying an unrestricted structure were excluded from the figures.
Structure  , DOI: ( /j.str )
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6. Figure 5
Influence of Cysteine Residues on the Allergenic Property of Art v 1
IgE-binding to Art v 1 and single cysteine mutants, i.e., C6S, C17S, C22S, C26S, C37S, C47S, C49S, and C53 was determined in ELISA using the allergic patient's serum previously applied for structural interaction studies. Measurements at 405 nm were performed in triplicates and presented as percentage of IgE reactivity to Art v 1 (= 100%).
Structure  , DOI: ( /j.str )
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7. Figure 6 IgE Interacting Amino Acids in Art v 1-Homologous Molecules
Defensin-proline domain fusions displaying more than 35% sequence homology with Art v 1 were identified in the following Asteraceae species: Ambrosia artemisiifolia (Leonard et al., 2010), Helianthus annuus (protein: P ), Cichorium endivia (EST: EL ), Senecio vulgaris (EST: DY ), Lactuca serriola (EST: DW ), and Taraxacum officinale (EST: DY ). Structures of homologous plant defensins without proline domain were found in Aesculus hipocastanum, Raphanus sativus, and Pisum sativum.
(A) Sequence alignment of Art v 1 and homologous proteins. Percentages of sequence identity to Art v 1 are given in parenthesis for each molecule.
(B) 3D structure of Art v 1 and homology modeling. The 3D structure models of proteins in ragweed, sunflower, groundsel, and lettuce were generated by homology modeling using the NMR structure of Art v 1 as template (RMS deviations: Ambrosia artemisiifolia 2.03 Å; Helianthus annuus 1.09 Å; Cichorium endivia 0.47 Å, Senecio vulgaris 0.55 Å; Lactuca serriola 0.48 Å; Taraxacum officinale 0.06 Å; Aesculus hipocastanum 2.35 Å; Raphanus sativus 2.40 Å and Pisum sativum 2.75 Å). Amino acids displaying chemical shift perturbations upon IgE interaction are colored in red for conserved residues, orange for residues with similar physicochemical properties, and yellow for residues that differ from those of the Art v 1 template. The boxed structures/models indicate molecules previously shown to bind IgE antibodies from Art v 1-sensitized patients.
Structure  , DOI: ( /j.str )
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